Biological sample preparation for testing

ABSTRACT

In one embodiment, a method for processing a sample includes selecting a selected sample from a biological specimen, the selected sample being in contact with a first surface of a first substrate. The method also includes transferring the selected sample directly from the first surface to a container comprising an internal volume. The method also includes forming or providing a sample solution within the internal volume of the container by contacting the selected sample with a lysis mixture using a protocol. The method further includes performing an assay, experiment, or test on the sample solution while the sample solution disposed is within the internal volume of the container. 
     In another embodiment, a method for processing a sample includes providing a selected sample comprising one or more cells. The method also includes transferring the selected sample into an internal volume of a container. The method also includes contacting the selected sample with a lysis mixture using a protocol to provide a sample solution, wherein the protocol comprises heating the sample solution to a first temperature that is greater than 37 degrees Celsius and less than or equal to 75 degrees Celsius.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/233,801, filed on Aug. 10, 2016. U.S. patent application Ser. No.15/233,801 claims the benefit of priority from U.S. Provisional PatentApplication No. 62/203,311, filed on Aug. 10, 2015, U.S. ProvisionalPatent Application No. 62/303,227, filed on Mar. 3, 2016, and U.S.Provisional Patent Application No. 62/341,563, filed on May 25, 2016.All applications cited in this section are incorporated by referenceherein; each in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present teachings generally relate to compositions, processes,methods, and kits for preparation of samples containing genetic materialfor downstream detection and/or quantitation analysis.

Description of the Related Art

Laser capture microdissection (LCM) is a technique for selecting ormicro dissecting specific cell(s) from a mixed population, usually undermicroscopic visualization. LCM techniques are used to isolate a singlecell or few cells from fresh or archived formalin-fixedparaffin-embedded (FFPE) tissues, blood, semen, or other biologicalsamples. Analysis of nucleic acids extracted using LCM from fresh orarchived formalin-fixed paraffin-embedded (FFPE) sections are used toprovide information about sample DNA (e.g., genotype) and RNA (e.g.,gene expression), for example, as they relate to sample morphologyand/or disease state. Using LCM, the biological sample may be examinedusing a microscope system and individual cells or group of cells may beselected and microdissected using one or more lasers. Through variousLCM techniques, the selected sample may be separated from unwantedportions of the larger sample and transferred to a container, such as awell, vial, or tube, for preparing the selected sample for use in adownstream assay, experiment, or test, for instance, in a polymerasechain reaction (PCR) or sequencing assay or workflow.

One objective in many LCM applications is to provide a large yield ofquality RNA from fresh or archived FFPE samples. The formalin fixing ofan FFPE sample can induce molecular cross-linking within samples, whichcomplicates retrieval of nucleic acids, as well as reduces their yieldand quality. This affects the efficiency of downstream detection.

In addition to increasing quality and yield of selected samples fromfresh or FFPE specimens, there exists a demand within the field toincrease total throughput by reducing the amount of time needed toprepare samples for downstream testing. Existing FFPE RNA Isolation Kitsrecommend a lengthy overnight Proteinase K (ProK) digestion at 37degrees Celsius for sample lysis, followed by several purification stepsto retrieve RNA from LCM FFPE tissue prior to downstream analysis. Thisapproach also requires sample transfer steps that can introduceadditional variability in the yield and quality of the RNA. Thismulti-step workflow can be especially challenging for RNA isolation fromlow-input sample types, such as single cells and small populations ofrare cells. Consequently, there is a need for an improved LCM FFPElysis-based solution for extraction of RNA from limited LCM FFPE samplesthat maintains RNA integrity that increases yield for downstreamapplications, for example, for PCR or sequencing, such as nextgeneration sequencing (NGS).

Therefore, new methods and systems that aid in the recovery of the highquality RNA from low input LCM-derived FFPE tissue material are highlydesirable, as well as improved ways of processing such samples in amanner that reduces processing time and reduces or eliminates transferof a selected sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention may be better understood from thefollowing detailed description when read in conjunction with theaccompanying drawings. Such embodiments, which are for illustrativepurposes only, depict novel and non-obvious aspects of the invention.The drawings include the following figures:

FIG. 1A is a schematic representation of a first system according toembodiments of the present invention.

FIG. 1B is a schematic representation of a second system according toembodiments of the present invention.

FIG. 2 is a schematic representation of a third system according toembodiments of the present invention.

FIG. 3 is a schematic representation of a fourth system according toembodiments of the present invention.

FIGS. 4A and 4B are schematic representations of two methods ofprocessing a selected sample using the system shown in FIG. 1B accordingto embodiments of the present invention.

FIGS. 5A and 5B are schematic representations of a method of processinga selected sample using the system shown in FIG. 3 according to anembodiment of the present invention.

FIG. 6 is a schematic representation of a method for processing aselected sample according to embodiments of the present invention.

FIG. 7 is a schematic representation of workflows according toembodiments of the current invention.

FIG. 8 is a schematic representation of a prior art LCM to PCR protocolworkflow (“Current Protocol”) and of an LCM to PCR protocol workflowaccording to an embodiment of the current invention (“New protocol”).

FIG. 9 is a bar graph showing RNA yield in terms of average Ct value forvarious lysis conditions or protocols.

FIG. 10 is a bar graph showing LCM FFPE isolated RNA qPCR performancewith Breast cancer FFPE tissue block>2 years old according based on twodifferent lysis conditions or protocols.

FIG. 11 is a schematic representation of a prior art LCM to sequencingprotocol (“Current Protocol”) and of an LCM to sequencing protocolworkflow according to an embodiment of the current invention (“Newprotocol”).

FIG. 12 is a bar graph showing RNA yield in terms of average Ct valuefor various lysis conditions according to the 1-day LCM to PCR protocolworkflow shown in FIG. 8.

FIG. 13 is an illustrative summary of a NGS RNA yield and Library datawith an Ampliseq Cancer 50 Panel.

FIGS. 14-16 are results from experiments for an RNA sample, useful todemonstrate advantages of embodiments of the present invention.

FIGS. 17-19 are results from experiments for a DNA sample, useful todemonstrate advantages of embodiments of the present invention.

FIG. 20 is an illustrative summary of a method according to anembodiment of the current invention.

FIGS. 21-24 are result from experiments useful to demonstrate advantagesof embodiments of the present invention

DETAILED DESCRIPTION OF THE DRAWINGS

As used herein, the term “contact” means in the state or condition oftouching or of immediate proximity Thus, if “A is in contact with B,then A and B are touching or in immediate proximity to one another.Contact between A and B does not necessarily mean that A and B arejoined, attached, or bonded to one another, such states are included.

Unless otherwise specified or obvious from its use, the term “about” or“approximately”, as used herein, means within ±10% of the statedquantity for which “about” or “approximately” is a modifier.

Referring to FIG. 1A, in certain embodiments of the present invention, asystem or instrument 100 a is schematically represented. System 100 amay be an optical system or instrument, for example, a microscope or alaser capture microdissection (LCM) instrument or apparatus, comprisingan optical head 102 a. System 100 a may further comprise a base 105 aconfigured to receive or support a substrate 110 a comprising a surface112 a containing, and/or in contact with, a biological specimen orheterogeneous mixture of biological cells 115 a. System 100 a may beconfigured for selecting, separating, isolating, and/or otherwiseproviding a selected or isolated sample 118 a from the biologicalspecimen or heterogeneous mixture of biological cells 115 a. Selectedsample 118 a may comprise a plurality of cells or may comprise a singlecell.

Optical head 102 a may include one or more lasers configured to removeselected sample 118 a from surface 112 a and/or to separate or isolateselected sample 118 a located on surface 112 a. In such embodiments, alaser or other means may be used levitate or otherwise transfer selectedsample 118 a into a cap, container, tube, capillary, or the like (notshown) for further processing and/or preparation. Selected sample 118 amay comprise a fresh sample (e.g., a fresh tissue sample), aformalin-fixed paraffin-embedded (FFPE) sample, or any other type ofbiological sample (e.g., any biological sample suitable for LCMprocessing).

Selected sample 118 a may be subsequently processed or prepared for adownstream assay, experiment, or test, for example, to detect, quantify,or characterize one or more deoxyribonucleic acid (DNA) molecules orsequences, or to detect, quantify, or characterize one or moreribonucleic acid (RNA) molecules or sequences.

Referring to FIG. 1B, in certain embodiments, a system or instrument 100b is schematically represented. System 100 b may be an optical system orinstrument, for example, a microscope or a laser capture microdissection(LCM) instrument, comprising an optical head 102 b. System 100 b mayfurther comprise a base 105 b configured to receive or support asubstrate 110 b comprising a surface 112 b containing, and/or in contactwith, a biological specimen or heterogeneous mixture of biological cells115 b. System 100 b may be configured for selecting, separating,isolating, and/or otherwise providing a selected or isolated sample 118b from the biological specimen or heterogeneous mixture of biologicalcells 115 b. Selected sample 118 b may comprise a plurality of cells ormay comprise a single cell.

Optical head 102 b may include one or more lasers configured to removeselected sample 118 b from surface 112 b and/or to separate or isolateselected sample 118 b located on surface 112 b. In such embodiments,gravity or other means, such as a laser, may be used to transferselected sample 118 b into a cap, container, tube, capillary, or thelike (not shown) for further processing and/or preparation. Selectedsample 118 b may comprise a fresh sample (e.g., a fresh tissue sample),a formalin-fixed paraffin-embedded (FFPE) sample, or any other type ofbiological sample (e.g., any biological sample suitable for LCMprocessing).

Selected sample 118 b may be subsequently processed or prepared for adownstream assay, experiment, or test, for example, to detect, quantify,or characterize one or more DNA molecules or sequences, or to detect,quantify, or characterize one or more RNA molecules or sequences.

Referring to FIG. 4A, a container, receptacle, tube, capillary, or thelike may be located below substrate 110 b to receive selected sample 118b once it is separated from surface 112 b. For example, a container 400shown in FIG. 4A may be located to receive selected sample 118 b as itis transferred via gravity or another external force, such as a forceinduced by a laser beam. In certain embodiments, a container 410includes a container cap 412, whereby selected sample 118 b may betransfer from substrate 110 b to the container cap 412 via gravity oranother external force such, as a force induced by a laser beam. Onceone or more selected samples 118 b have been transferred to containercap 412, the container cap may be inserted into an opening 415 ofcontainer 400 so that sample(s) 118 b may be received into container 400and processed further.

Referring to FIG. 2, in certain embodiments, a system or instrument 200is schematically represented. System 200 may be an optical system orinstrument, for example, a microscope or a laser capture microdissection(LCM) instrument, comprising an optical head 202. System 200 may furthercomprise a base 205 configured to receive or support substrate 206comprising a surface 207 and a substrate 210 comprising a surface 212containing. System 200 may be configured for selecting, separating,isolating, and/or otherwise providing a selected or isolated sample 218from a biological specimen or heterogeneous mixture of biological cells215. Selected sample 218 may comprise a plurality of cells or maycomprise a single cell.

Optical head 202 may include one or more lasers configured to removeselected sample 218 from surface 212 and/or to separate or isolateselected sample 218 located on surface 212. In such embodiments, system200 and surface 212 of substrate 210 may be configured such that, uponseparating substrate 210 from substrate 206, selected sample 218 remainsattached and/or in contact with surface 212 and the remaining portionsof biological specimen 215 remain attached and/or in contact withsurface 207. Selected sample 218 may comprise a fresh sample (e.g., afresh tissue sample), a formalin-fixed paraffin-embedded (FFPE) sample,or any other type of biological sample (e.g., any biological samplesuitable for LCM processing).

In certain embodiments, substrate 210 comprises one or more of a lasercapture microdissection cap (LCM cap), sample carrier, or extractiondevice. Exemplary systems, devices, and methods suitable to select,separate, isolate, and/or otherwise transfer one or more selected sample218 may be found, but are not limited to, those discussed in U.S. Pat.Nos. 5,859,699 7,075,640, 6,690,470, 8,346,483, 7,456,938, 8,722,357,6,528,248, 7,776,273, 7,229,595, 7,749,388, 6,469,779.

During use, each of the surfaces 207, 212 are in contact with thebiological specimen 215. System 200 may be configured to select,separate, isolate, and/or otherwise transfer selected sample 218 fromsurface 207 of substrate 206 to surface 212 of substrate 210. Substrate210 may be lifted or removed from substrate 206 so that selected sample218 can be separated from biological specimen 215 for further processingand/or used in an assay, experiment, or test in order to characterize orquantify the content of selected sample 218. For example, selectedsample 218 may be subsequently processed or prepared for a downstreamassay, experiment, or test, for example, to detect, quantify, orcharacterize one or more DNA molecules or sequences, or to detect,quantify, or characterize one or more RNA molecules or sequences.

Referring to FIG. 3 and FIG. 5A, in certain embodiments, a substrate 310comprises an LCM cap 310, where surface 312 of LCM cap 310 may have adiameter configured for attachment of LCM cap 310 to a relatively smallcontainer 500 (e.g., a container or vial having an internal volume of0.2 milliliters or about 0.2 milliliters). Embodiments of LCM cap 310suitable for embodiments of the current invention are discussed in U.S.Provisional Application No. 62/203,311. In certain embodiments, system200 may be configured for use with both a LCM cap 210 (e.g., for usewith containers or vials of a relatively large volume) and LCM cap 310(e.g., for use with containers or vials of a relatively large volume andvials of a relatively small volume). In such embodiments, LCM cap 210may be configured to receive a container or vial having a volume of 0.5milliliters, while LCM cap 310 is configured to receive a container orvial having a volume of 0.5 milliliters or about 0.5 milliliters and acontainer or vial having a volume of 0.2 milliliters or about 0.2milliliters.

The use an LCM cap 310 with a container 500 having a volume of 0.2milliliters or about 0.2 milliliters has been discovered to provideparticular advantages in processing certain types of selected samples218. For example, a 0.2 millimeter container or vial may be used toprocess selected samples comprising only a few cells or containing onlya single cell. As discussed in greater detail below, use of a 0.2millimeter container or vial may be used in conjunction with LCM cap 310to advantageously process the selected sample (e.g., by a lysis assay)and, in the same container or vial, perform a downstream assay,experiment, or test. In the context of selected samples (e.g., selectedsample 218) comprising only a few cells or a single cell, the use of asingle container advantageously helps to preserve the sample solutionand reduce contamination that can occur when a sample solution istransferred from contain or vial to another.

For example, referring to FIG. 5A, in certain embodiments, LCM cap 310is separated from substrate 206 (top left of FIG. 5A) and moved to acontainer 500 comprising an internal volume 505 and containing, forexample, a volume 508 of a lysis mixture 510. The smaller diameter atsurface 212 of LCM cap 310 may subsequently be lowered or fitted ontocontainer 500 (center of FIG. 5A) to provide a closed container 502. Incertain embodiments, closed container 502 may be inverted so that lysismixture 510 contacts selected sample 218 to form a sample solution 512(bottom left of FIG. 5A). With additional reference to FIG. 5B, LCM cap310 and container 500 may be placed on a holder, platform, or rack 520.Holder 520, including LCM cap 310 and container 500, may be placedinside an incubator 525 for incubation of selected sample 218. In someembodiments, a plurality of LCM cap 310/container 500 pairs having thesame or similar geometry are provided, for example, where each LCM cap300 contains a respective selected sample 218 provided by system 200 ortwo or more such systems. In such embodiments, some or all of the LCMcaps 310 may be placed on holder 520 and/or be simultaneously placed inincubator 525 for incubation of the selected samples.

In certain embodiments, container 500 comprises a well of a microtiterplate (e.g., of a 96 well microtiter plate or a 384 well microtiterplate) and LCM cap 310 is placed or fitted onto an opening at the top ofthe well. Additional LCM caps 310, each containing a selected sample218, may be placed on different ones of the wells of the microtiterplate (e.g., on adjacent wells of the microtiter plate or on wellsseparated by an intermediate well). Once placed on the microtiter plate,each selected sample 218 may be prepared or processed as discussedfurther herein (e.g., the selected samples 218 may together be placed inincubator 525, for example, to perform a lysis assay on samples 218).

Additionally or alternatively, other processing of selected sample 218(alone or with additional samples 218) may be performed within volume asdiscussed in greater detail below. During processing of selected sample218 within the volume 505 that is defined by LCM cap 310 and container500, LCM cap 310 may be removed and re-attached as need (as indicated bythe double arrow in center of FIG. 5A) to add other reagents orsolutions as prescribed or needed. Once preparation and processing ofsample 218 is completed, LCM cap 310 may be removed, and sample 218 maybe placed into or attached to an instrument 520 for performing adownstream assay, experiment, or test on sample solution 512, which nowcontains the processed version of the initially selected sample 218.Advantageously, the entire process, from obtaining the selected sample218 to performing an assay, experiment, or test on the processed samplesolution 512, takes place with selected sample 218 confined to surface312 of LCM cap 310 and/or volume 505 of container 500.

The above example of processing and preparing a selected was primarilywith reference to system 200 and LCM cap 310 substrate. However, it willbe appreciated that at least portions of the previous paragraphs may beapplied to systems 100 a, 100 b and to substrates 110 a, 110 b, and 210for preparing selected samples such as selected samples 118 a or 118 b.For example, a selected sample (e.g., selected sample 118 a or 118 b, orselected sample 218 selected using substrate 210) and a lysis mixturemay be brought into contact with one another when (e.g., as discussedabove in reference to FIGS. 1, 4A, and 4B) a selected sample is lowered,falls, or is levitated into a container.

In the previous embodiment using LCM cap 310 or in embodiments based onuse of substrates 110 a, 110 b, or 210, a lysis mixture may be added tothe container (e.g., container 400, 500 and/or container cap 412) whenthe selected sample is so collect. Alternatively, the lysis mixture maybe added to the container prior to collection of the selected sample.The lysis mixture may comprise a buffer system configured to provide adirect lysate to qPCR workflow. Additionally or alternatively, the lysismixture may comprise a buffer system configured to provide a directlysate to sequencing assay workflow.

Referring to FIG. 6, in certain embodiments, a method 600 for processinga selected sample (e.g., selected samples 118 a, 118 b, or 218)containing (e.g., in container 400, 500) one or more nucleic acidsincludes an element 610, which comprises providing a selected samplecomprising one or more biological cells. Method 600 further includes anelement 620, comprising placing, locating, or transferring the selectedsample into an internal volume of a container (e.g., as discussed abovein relation to selected samples 118 a, 118 b, or 218 into, for example,internal volume 505 of container 500 or the internal volume of container400).

Method 600 also includes element 630, comprising contacting the selectedsample with a lysis mixture (e.g., lysis mixture 510) using a protocolto form or provide a sample solution (e.g., the sample solution 512formed by selected sample 218 and lysis mixture 510) within the internalvolume of the container. The protocol may include reagents andprocedures for lysing the selected sample. As discussed below, theprotocol may also include other reagents and procedures for processingand preparing the selected sample for downstream assays, experiments, ortests. The lysis mixture may comprise one or more of:

-   -   A low ionic strength detergent-based buffer system.    -   A Proteinase K.    -   A lysis mixture has a volume that is less than or equal to 10        microliters.

Exemplary formulation for lysis mixtures include, but are not limitedto, those discussed in U.S. Pat. No. 7,964,350. Embodiments in which thelysis mixture volume is 10 microliter or less have been found to beuseful where the selected sample contains only one or a few cell and/orin combination with an container (e.g., container 400 or 500) have avolume of about 0.2 milliliters or less. In such embodiments, a greatbenefit has been discovered doing all processing in a single container.

In certain embodiments, the protocol according to method 600 comprisesheating the sample solution (e.g., sample solution 512) to a firsttemperature that is greater than 37 degrees Celsius and less than orequal to 75 degrees Celsius. As discovered during experiments discussedbelow and shown in FIGS. 9, 10, 12, and 14-18, lysis assays conducted ata temperature above 37 degrees Celsius were found to increase yield of atarget DNA or RNA from a selected sample. This was demonstratedexperimentally by a lower Ct value (e.g., average number of thermalcycles in a qPCR amplification process) for assays conducted attemperatures above 37 degrees Celsius as compared to the Ct values ofassays conducted at 37 degrees Celsius. In certain embodiment, it wasdiscovered that the yield for both DNA and RNA was higher at lysis assaytemperature of 65 degrees Celsius or at about 65 degrees Celsius (e.g.,65 degrees Celsius±1 degrees Celsius) than at temperatures below orabove this temperature condition. In certain sets of lysis protocolsconducted, a lysis temperature greater than or equal to 45 degreesCelsius, or 55 degrees Celsius, and less than or equal to 75 degreesCelsius was found produce a better DNA and/or RNA yield than when thelysis assay was conducted either above or below these temperatureconditions.

In certain embodiments, the protocol of element 630 of method 600comprises a first incubation for assaying the selected sample that isperformed over a first incubation period and at the first temperature(e.g., at a temperature of about 65 degrees Celsius). The firstincubation period is less than or equal to 2 hours or less than or equalto 1 hour. Experiment according to embodiments of the present inventionas discussed below were generally conducted over an incubation period ofabout 1 hour and provided the advantage of increased DNA and RNA yields.Lower incubation periods advantageously allow more target samples to beprocessed over a given period of time.

In certain embodiments, the protocol according to method 600 may includean element 640, comprising exposing the sample solution (e.g., samplesolution 512) to second temperature that is greater than the firsttemperature, for example, at a temperature of at least 85 degreesCelsius. Element 640 may further comprise a second incubation at theelevated temperature that follows the first incubation, wherein thesecond incubation period is at least 15 minutes or about 15 minutes(e.g., 15 minutes, 30 minutes, 45 minutes, or 60 minutes). The durationof the second incubation may be selected to balance demands of increaseyield and higher processing throughput for a particular application. Incertain embodiments, the second incubation at the higher temperature maybe used to decreases a molecular crosslinking of nucleic acid moleculeswithin the sample solution.

In some embodiments, method 600 additionally includes an element 650,comprising adding a deoxyribonuclease (DNase) and/or a ribonuclease(RNase) to the sample solution (e.g., sample solution 512), which mayresult in a modified or larger sample solution (e.g., sample solution512). In some embodiments, the temperature of the solution is loweredfrom the first temperature (used during lysing), either before or afteraddition of the DNase and/or RNase. For example, the solutiontemperature may be lower to a temperature below 40 degrees Celsius(e.g., to a temperature the is at 37 degrees Celsius or is about 37degrees Celsius or a temperature that is below 37 degrees Celsius, forexample, to room temperature). In certain embodiments, the samplesolution comprises both a DNA molecule and an RNA molecule and differentportions of the solution are treated with either RNase or DNase. In suchembodiments, the solution within the container (e.g., container 400 orcontainer 500) may be divided to provide a first portion of the samplesolution remaining the container and portion transferred (e.g., pouredor pipetted) to a second container. An RNase reagent may be added toeither the first portion or the second portion, wherein DNA assay,experiment, or test may be performed on that portion. A DNase reagentmay be added to the other portion so that an RNA assay, experiment, ortest may be performed on that portion. In certain embodiments, the atleast a portion of the first and/or second portions may be transferredto a third container for the same sample preparation and/or downstreamassay, test, or experiment as the first or second portions.Alternatively, the third portion may be processed differently than thefirst or second portions and/or be included in a different downstreamassay, test, or experiment than either the first or second portions.

In certain embodiments, method 600 may further include an element 660,comprising adding a stop solution to the sample solution (e.g., samplesolution 512). In the case of RNA samples, method 600 may also includean element 670 comprising adding a reverse transcriptase to the samplesolution (e.g., for converting RNA molecules to cDNA molecules). Method600 may additionally include an element 670, comprising performing apre-amplification assay on the sample solution prior to performing theassay, experiment, or test on the sample solution. In some embodiments(e.g., where there is only small amount or number of a particular typeof molecule, such as a RNA or DNA target molecule), method 600 mayinclude an element 680, comprising performing a preamplification assayon the sample solution prior to performing an assay, experiment, or teston the sample solution for detection, analysis, quantification of one ormore molecules from the separated sample. Examples of solutions andassays for processing the selected sample include, but are not limitedto, those discussed in U.S. Pat. No. 7,964,350.

In various embodiments, method 600 may include an element 690,comprising performing one or more assays, experiments, or tests in thesame container (e.g., container 400 or 500) used to initially lyse theselected sample (e.g., selected sample 118 a, 118 b, or 218). The assay,experiment, or test may include one or more of:

-   -   A polymerase chain reaction (PCR) assay, experiment, or test.    -   A quantitative PCR (qPCR) assay, experiment, or test.    -   A digital PCR assay, experiment, or test.    -   A genotyping assay, experiment, or test.    -   A sequencing assay, experiment, or test.

The sequencing assay, experiment, or test may comprise capillaryelectrophoresis assay, experiment, or test or next generation sequencingassay, experiment, or test. The next generation sequencing assay,experiment, or test comprises one or more of:

-   -   A single-molecule sequencing assay, experiment, or test.    -   An Ion semiconductor assay, experiment, or test.    -   A pyrosequencing assay, experiment, or test.    -   A sequencing by synthesis assay, experiment, or test.    -   A sequencing by ligation assay, experiment, or test.    -   A chain termination assay, experiment, or test.    -   A Sanger sequencing assay, experiment, or test.

In certain embodiments, a sample solution may comprise a firstpopulation of a first molecule and a second population of a raremolecule, wherein the second population is less than the firstpopulation. In such embodiments, the assay, experiment, or testcomprises an assay, experiment, or test for detecting, analyzing, orquantifying the rare molecule.

One or more of the above elements of method 600 may be omitted ormodified. For example, element 670 (adding a reverse transcriptase) maybe omitted if a DNA assay, experiment, or test are to be conducted onthe sample solution. In other embodiments, element 640 (processing at atemperature above the nominal lysing temperature) may be omitted. In yetother embodiment element 690 may be omitted and all or a portion of thesample solution may be transferred to a different container for adownstream assay, experiment, or test.

EXAMPLES

Support for at least some of claimed and/or disclosed embodiments of thecurrent invention are supported by various experiments and testsdiscussed below and found in FIGS. 7-18.

Disclosed workflows disclosed and/or experimentally tested combine threecomponents that may be used in various combinations to allow or provideefficient lysis and/or extraction of RNA from LCM captured fresh and/orFFPE tissue/cell lysate:

1) LCM cap was configured to be attached to low volume containers (e.g.,container have a volume of about 0.2 milliliters or less) (see U.S.Provisional Application No. 62/203,311 for suitable embodiments of anLCM cap).

2) Buffer formulations for direct FFPE lysis in a small volume (e.g.,about 0.2 milliliter container volume and/or a lysis mixture volume thatis less than or equal to 10 microliters or about 10 microliters),

3) Predetermined lysis conditions for FFPE (incubation temperature andtime) (e.g., FIG. 7).

Together, these components enable an extraction workflow to be completedin a single collection tube. Moreover, these elements are compatiblewith a single-tube qPCR workflow (FIG. 8).

For example FIG. 9 shows results for an LCM cap used in combination witha small volume container (0.2 milliliter) using LCM FFPE isolated RNAqPCR performance with Specific NSCLC targets (Lung Cancer FFPE tissueblock>1 year old).

The LCM cap compatible with a 0.2 ml PCR container was used to processLCM FFPE cell/tissue isolates—from sample collection throughdetection—in a single tube, which minimizes sample losses associatedwith multi-tube workflows (for example, a LCM cap compatible with a 0.2milliliter container). In addition, the flexibility to use a small lysisvolume (10 microliter) enables our customers to increase sampleconcentration as well sensitivity for downstream workflows, which isespecially beneficial when working with very small LCM samples such assingle cells. Our lysis based solution workflow leverages the Cells toCt buffer formulation, which is a low ionic strength detergent-basedbuffer system suitable for direct lysate to qPCR workflow. Manyliteratures have sited that the yield and recovery of RNA gets betterwith higher temperature and short incubation. In this proposed workflowthe cell lysis condition was achieved by heating the micro-dissected LCMFFPE cells or tissue lysate at 65 degrees Celsius for <1 hour instead of37 degrees Celsius overnight (16 hours). Additional advantage of lysingthe LCM micro dissected cells at 65 degrees Celsius is to enablemulti-analyte unified workflow to extract DNA and RNA from same LCMtissue lysate. Alternatively, the proposed workflow can be used toisolate RNA and genomic DNA alone from LCM FFPE tissue lysate.Increasing the temperature of cell lysis condition from 37 degreesCelsius to 65 degrees Celsius has shown improvements in RNA yield (FIG.9). Since FFPE samples have substantial RNA crosslinking that couldaffect the efficiency of the RT enzyme, we recommend heating the samplefor 15 mins at 85 degrees Celsius after cell lysis step to improvemolecular de-crosslinking Incubation time optimization study with the 85degrees Celsius step (15 minutes, 30 minutes, and 1 hour) indicated nostatistical significant difference between samples.

Referring to FIG. 9, LCM tissue slides were prepared from Lung (NSCLC)block>1 year old and Breast cancer FFPE block>2 year old. Preliminaryworkflow feasibility was achieved by performing RT-qPCR with cancerspecific assays tested with RNA obtained from LCM tissue lysateincubated at 37 degrees Celsius overnight and our improved LCM FFPEtissue RNA extraction protocol (65 degrees Celsius 1 hour and 85 degreesCelsius 15 mins-1 hr). Proposed LCM FFPE cell extraction workflow (65degrees Celsius 1 hr and 85 degrees Celsius 15 mins-1 hr) indicatedbetter/comparable performance with respect to existing (37 degreesCelsius—overnight) protocol for majority of assays (control and target)tested in this study (FIG. 9 and FIG. 10). The improved workflowaccording to an embodiment of the present invention took advantage of anLCM cap compatible with a 0.2 milliliter container, offering low volumeflexibility, and providing compatibility with a single-tube qPCRworkflow. Feasibility data with improved cell extraction protocolindicated successful extraction of qPCR and NGS grade RNA frommicro-dissected LCM-FFPE cells or tissue with a shorter turnaround time(>80%) compared to current (37 degrees Celsius—overnight) cellextraction protocol, which is highly desirable for modern genomicanalysis.

Referring to FIG. 10, a study was performed using FFPE 7 micron sectionfrom a year old NSCLC FFPE block and qPCR analysis was performed on RNAsamples processed from a 200-micron FFPE region micro dissected usingLCM cap compatible with a 0.2 ml PCR tube. Optimized FFPE—RNA 2 hourslysis protocols indicated comparable result compared to current 37degrees Celsius O/N (16 hours) incubation. All control and targetedassays tested for NSCLC lung carcinoma are detected with both ourcurrent FFPE RNA extraction method as well as the improved one. Each barrepresents duplicate sample preparations and quadruple PCR replicates.

Referring to FIGS. 11 and 12, a study was performed using FFPE 7 micronsection from a >2 year old Breast FFPE block and qPCR analysis wasperformed on RNA samples processed from a 150, 500 and 4000-micron FFPEregion micro dissected using an LCM cap compatible with a 0.2 ml PCRtube. Optimized FFPE—RNA<than 2 hours lysis protocols indicatedcomparable/better result with respect to current 37 degrees Celsiusovernight (16 hours) incubation. Each bar represents triplicate samplepreparations and quadruple PCR replicates.

Referring to FIGS. 14-16, results of RNA qPCR experiments aredemonstrated:

-   -   FFPE breast tissue lysate was incubated at temperature mentioned        in the table.    -   Lysate buffer used: Cells to Ct buffer    -   After 1 hour incubation reaction (for each time point) Lysate        was treated with DNase    -   Reaction was stopped by adding stop solution    -   Sample was assessed by RT-PCR assay

Referring to FIGS. 17-19, results of DNA qPCR experiments aredemonstrated:

-   -   FFPE breast tissue lysate was incubated at temperature mentioned        in the table.    -   Lysate buffer used: Cells to Ct buffer    -   After 1 hour incubation reaction (for each time point) Lysate        was treated with RNase    -   Reaction was stopped by adding stop solution    -   Sample was assessed by PCR assay

Regarding FIGS. 16 and 19:

-   -   Boundary test for 65 C: Sample lysates were tested at ±1 C (64 C        and 66 C).    -   Tukey Kramer analysis indicated that there is no significant        difference between 64 C, 65 C and 66 C for both DNA and RNA        yield. (circles overlapping—no significant difference)

The above presents a description of the best mode contemplated ofcarrying out the present invention, and of the manner and process ofmaking and using it, in such full, clear, concise, and exact terms as toenable any person skilled in the art to which it pertains to make anduse this invention. This invention is, however, susceptible tomodifications and alternate constructions from that discussed abovewhich are fully equivalent. Consequently, it is not the intention tolimit this invention to the particular embodiments disclosed. On thecontrary, the intention is to cover modifications and alternateconstructions coming within the spirit and scope of the invention asgenerally expressed by the following claims, which particularly pointout and distinctly claim the subject matter of the invention.

The following United States patents are all herein incorporated byreference in their entirety:

U.S. Pat. No. 5,859,699; U.S. Pat. No. 6,157,446; U.S. Pat. No.6,469,779; U.S. Pat. No. 6,495,195; U.S. Pat. No. 6,528,248; U.S. Pat.No. 6,690,470; U.S. Pat. No. 6,887,703; U.S. Pat. No. 7,049,558; U.S.Pat. No. 7,075,640; U.S. Pat. No. 7,229,595; U.S. Pat. No. 7,456,938;U.S. Pat. No. 7,473,401; U.S. Pat. No. 7,556,733; U.S. Pat. No.7,749,388; U.S. Pat. No. 7,776,273; U.S. Pat. No. 7,964,350; U.S. Pat.No. 8,288,106; U.S. Pat. No. 8,346,483; U.S. Pat. No. 8,715,955; U.S.Pat. No. 8,722,357; U.S. Pat. No. 8,828,664; U.S. Pat. No. 9,279,152.

What is claimed is:
 1. A method for processing a tissue sample,comprising: dissecting with an optical system cell sample comprising oneor more biological cells from a formalin-fixed, paraffin embedded (FFPE)tissue sample; transferring the sample of one or more biological cellsinto an internal volume of a container; extracting the cell sample witha lysis mixture using an extraction protocol, wherein the extractionprotocol comprises: incubating the cell sample with the lysis mixture ata first temperature of about 64° C. to about 66° C. for one hour;increasing the incubation temperature of the cell sample to a secondtemperature of about 85° C. for at least 15 minutes; and stopping lysisof the cell sample, thereby providing a sample solution containing oneor more nucleic acids in the container.
 2. A method for processing asample containing one or more nucleic acids, comprising: providing aselected sample comprising one or more biological cells; transferringthe selected sample into an internal volume of a container; contactingthe selected sample with a lysis mixture using a protocol to provide asample solution; wherein the protocol comprises heating the samplesolution to a first temperature that is greater than or equal to 45degrees Celsius and less than or equal to 75 degrees Celsius.
 3. Themethod according to claim 2, further comprising performing an assay,experiment, or test on the sample solution.
 4. The method according toclaim 1, wherein before stopping lysis of the cell sample, the methodfurther comprises preparing an RNA sample solution by adding DNase to atleast part of the sample solution.
 5. The method according to claim 4,wherein after stopping lysis of the cell sample, the method furthercomprises performing reverse transcription on at least part of the RNAsample solution to form a cDNA sample solution.
 6. The method of claim5, wherein the method further comprises performing an assay, experiment,or test on the cDNA sample solution.
 7. The method of claim 6, whereinperforming an assay, experiment, or test on the cDNA sample solutioncomprises sequencing the cDNA sample solution.
 8. The method of claim 6,wherein performing an assay, experiment, or test on the cDNA samplesolution comprises performing a polymerase chain reaction (PCR) assay,experiment or test on the cDNA sample solution.
 9. The method accordingto claim 5, wherein the method further comprises pre-amplifying at leastpart of the cDNA sample solution to form a pre-amplified cDNA samplesolution.
 10. The method of claim 9, wherein the method furthercomprises performing an assay, experiment, or test on the pre-amplifiedcDNA sample solution.
 11. The method of claim 10, wherein performing anassay, experiment, or test on the pre-amplified cDNA sample solutioncomprises sequencing the pre-amplified cDNA sample solution.
 12. Themethod of claim 10, wherein performing an assay, experiment, or test onthe pre-amplified cDNA sample solution comprises performing a polymerasechain reaction (PCR) assay, experiment or test on the pre-amplified cDNAsample solution.
 13. The method according to claim 1, wherein afterstopping lysis of the cell sample, the method further comprisespreparing a DNA sample solution by pre-amplification of least part ofthe sample solution.
 14. The method of claim 13, wherein performing anassay, experiment, or test on the DNA sample solution comprisessequencing the DNA sample solution.
 15. The method of claim 14, whereinperforming an assay, experiment, or test on the DNA sample solutioncomprises sequencing the DNA sample solution.
 16. The method of claim14, wherein performing an assay, experiment, or test on the DNA samplesolution comprises performing a polymerase chain reaction (PCR) assay,experiment or test on the DNA sample solution.
 17. The method of claim3, wherein performing an assay, experiment, or test on the samplesolution comprises sequencing the sample solution.
 18. The method ofclaim 3, wherein performing an assay, experiment, or test on the samplesolution comprises performing a polymerase chain reaction (PCR) assay,experiment or test on the sample solution.